Identification of fundamentally cytoprotective pathways in the myocardium lingers as an elusive challenge. Although ischemic preconditioning may be the most powerful experimental cardioprotective strategy known, little clinical application has resulted from its discovery. Based on the premise that transient metabolic alterations occur during ischemic preconditioning, the PI has developed a novel hypothesis wherein activation of a metabolic signal exerts protective effects. Emerging evidence indicates that global increases in levels of a specific serine/threonine post-translational modification, beta-O-linked N-acetylglucosamine (O-GlcNAc), protects cell lines against injury. Here, the PI will attempt to establish this modification as a novel cardiac survival signal and elucidate its potential involvement in myocardial ischemia-reperfusion injury. The general structure of this proposal is designed to answer the questions: What happens to O-GlcNAc levels during ischemia-reperfusion and preconditioning? Is the O-GlcNAc signal sufficient for cardioprotection? What are the target(s) in this process? Does this potentially protective pathway result in chronic improvement in cardiac function? In Specific Aim I (SA I), the applicant will identify temporal changes in O-GlcNAc levels during myocardial ischemia-reperfusion and following ischemic preconditioning. Using complementary pharmacology, adenoviral gene transfer, and RNA interference-mediated translational silencing, the PI will evaluate whether augmentation of O-GlcNAc levels attenuates cardiac injury following in vivo ischemia or in vitro hypoxia-reoxygenation (SA II). Next, the applicant will characterize the downstream targets, namely in the mitochondria (e.g. KATP), of O-GlcNAc-mediated protection using isolated myocytes exposed to hypoxia-reoxygenation and in vivo myocardial ischemia-reperfusion (SA III). Lastly, the PI will ascertain whether acute O-GlcNAc-mediated protection improves long-term cardiac function in vivo following myocardial infarction (SA IV). Data garnered from such innovative studies may establish a novel paradigm in the mechanism of post-ischemic cardiac injury and protection. Possible findings during this project will allow the development of new strategies to combat acute myocardial infarction in patients. LAY SUMMARY: The applicant will focus on a poorly understood metabolic pathway to evaluate the mechanisms of injury during a heart attack. Information gathered from this proposal may help treat heart disease.